Hp. Meissner

Significance of ionic fluxes and changes in membrane potential for stimulus-secretion coupling in pancreatic B-cells

This brief review has tried to shed some light on the mechanisms and significance of the changes in membrane potential and in ionic fluxes occurring in B-cells upon glucose stimulation. There is now strong evidence that, under physiological conditions at least, these electrical events-and the underlying modifications of ionic permeabilities and fluxes — play a causal role in the stimulation of insulin release. It also seems clear that certain accompanying ionic fluxes have no direct stimulatory role, but may be important in maintaining cellular homeostasis. Recent experimental evidence has also shown that the electrical activity in B-cells is not an all-or-none stereotypic response. Not only can its intensity be adjusted to the magnitude of the stimulus, but its characteristics can also be modulated by potentiators Our knowledge of the stimulus-secretion coupling has markedly progressed over the past few years, but elucidation of several important steps remains a challenging goal. There is no doubt that parallel measurements of insulin release, of ionic fluxes and of membrane potential in B-cells will still contribute to that understanding.

Mechanism of the stimulation of insulin release in vitro by HB 699, a benzoic acid derivative similar to the non-sulphonylurea moiety of glibenclamide

SummaryHB 699 is a benzoic acid derivative similar to the non-sulphonylurea moiety of glibenclamide. The mechanisms whereby it affects B-cell function have been studied in vitro with mouse islets. In the presence of 3 mmol/l glucose, HB 699 decreased 86Rb+ efflux and accelerated 45Ca2+ efflux from islet cells, depolarized the B-cell membrane and induced an electrical activity similar to that triggered by stimulatory concentrations of glucose, and increased insulin release. The changes in 45Ca2+ efflux and insulin release, but not the inhibition of 86Rb+ efflux, were abolished in the absence of Ca2+. In the presence of 10 mmol/l glucose, HB 699 increased 86Rb+ and 45Ca2+ efflux from the islets, caused a persistent depolarization of the B-cell membrane with continuous electrical activity, and markedly potentiated insulin release. All these changes were suppressed by omission of extracellular Ca2+. In the presence of 15 mmol/l glucose, diazoxide increased 86Rb+ efflux, hyperpolarized the B-cell membrane, suppressed electrical activity and inhibited insulin release. HB 699 reversed these effects of diazoxide. It is suggested that HB 699 decreases K+ permeability of the B-cell membrane, thereby causing a depolarization which leads to activation of voltage-dependent Ca channnels and Ca2+ influx, and eventually increases insulin release. A sulphonylurea group is thus not a prerequisite to trigger the sequence of events that is also thought to underlie the releasing effects of tolbutamide and glibenclamide.

9-Aminoacridine- and tetraethylammonium-induced reduction of the potassium permeability in pancreatic B-cells. Effects on insulin release and electrical properties.

The effects of 9-aminoacridine and tetraethylammonium on insulin release and rubidium efflux from perifused rat islets were investigated and correlated with their effects on the electrical properties of mouse B cells studied with microelectrode techniques. 9-Aminoacridine (0.05--1 mmol/l) and tetraethylammonium (2--40 mmol/l) produced a dose-dependent, reversible potentiation of glucose-stimulated insulin release. This effect was rapid, affected both phases of secretion and was maximum in the presence of 6 mmol/l glucose, but no longer significant at 20 mmol/l glucose. It was unaltered by atropine or propanolol, and abolished by mannoheptulose or omission of extracellular calcium. 9-Aminoacridine, but not tetraethylammonium, also induced insulin release in the absence of glucose stimulation. Neither drug modified glucose metabolism in islet cells and only 9-aminoacridine increased 45Ca2+ uptake. In the presence of 0, 3 or 6 mmol/l glucose, but no longer at 20 mmol/l glucose, 9-aminoacridine and tetraethylammonium reduced the rate of 86Rb+ efflux from the islets. Both drugs also slightly reduced 86Rb+ uptake by islet cells. In the presence of 11 mmol/l glucose, 9-aminoacridine reduced the amplitude and the duration of the polarization phases between the bursts of electrical activity; concomitantly these periods of spike activity were markedly prolonged. At lower glucose concentrations (3 or 7 mmol/l), 9-aminoacridine progressively depolarized B cells and induced electrical activity in otherwise silent cells. Tetraethylammonium also suppressed the repolarization phases between the bursts of spikes in the presence of a stimulating concentration of glucose. At low glucose, tetraethylammonium produced only a limited and not maintained depolarization. These results show that a reduction of the potassium permeability in pancreatic B cells potentiates the insulin-releasing effect of glucose and may even stimulate secretion. They also suggest that the initial depolarizing effect of glucose is due to a reduction of the potassium permeability, whereas the repolarization at the end of each burst of electrical activity is mediated, at least in part, by an increase in the potassium permeability of B cells.

Effects of theophylline and dibutyryl cyclic adenosine monophosphate on the membrane potential of mouse pancreatic beta‐cells.

The effects of theophylline and dibutyryl cyclic AMP on the membrane potential of mouse beta‐cells were studied with micro‐electrodes. They were compared to their effects on insulin release by perifused mouse islets. In 3 mM‐glucose, theophylline (10 mM) depolarized the beta‐cell membrane and stimulated insulin release, but did not induce electrical activity. Dibutyryl cyclic AMP (1 mM) was without effect. In 7 mM‐glucose, theophylline (0.5‐2 mM) and dibutyryl cyclic AMP (1 mM) slightly depolarized the beta‐cell membrane, induced electrical activity in otherwise silent cells and increased insulin release. A higher concentration of theophylline (10 mM) hyperpolarized the beta‐cell membrane, did not induce electrical activity, but also stimulated insulin release. In 10 mM‐glucose, the membrane potential of beta‐cells exhibited repetitive slow waves with bursts of spikes on the plateau. Under steady state, these slow waves were differently affected by low or high concentrations of theophylline. At 0.5‐2 mM, theophylline shortened the intervals, lengthened the slow waves and slightly increased their frequency. On the other hand, 10 mM‐theophylline markedly decreased the duration of both intervals and slow waves, and increased their frequency. The effects of 1 mM‐dibutyryl cyclic AMP were similar to those of 2 mM‐theophylline. With 2‐10 mM‐theophylline, two other effects were also observed: a transient hyperpolarization with suppression of electrical activity immediately after addition of the methylxanthine and an increase in electrical activity upon its withdrawal. Theophylline and dibutyryl cyclic AMP markedly potentiated insulin release induced by 10 mM‐glucose. The magnitude of these changes did not correlate well with the importance of the changes in electrical activity. However, with 2‐10 mM‐theophylline the increase in release was also preceded by an initial transient inhibition, whereas withdrawal of the methylxanthine was accompanied by a further increase. When Ca influx was inhibited by D600, the slow waves were suppressed, the membrane was depolarized to the plateau level and only few spikes were present. Although theophylline markedly increased insulin release under these conditions, it did not affect the membrane potential. Several conclusions can be drawn from this study. Insulin release and electrical activity in beta‐cells can be dissociated when intracellular Ca is used to trigger exocytosis. High concentrations of theophylline produce effects unrelated to cyclic AMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of a Calcium-channel Agonist On the Electrical, Ionic and Secretory Events in Mouse Pancreatic B-cells

The changes in pancreatic B-cell function produced by a Ca channel agonist, the dihydropyridine derivative CGP 28392, have been studied with mouse islets. CGP 28392 (5 microM) modified the electrical activity induced in B-cells by 10 mM glucose: the duration and the amplitude of the slow waves of membrane potential increased, but the overall spike activity decreased. Simultaneously, CGP 28392 markedly increased insulin release and 45Ca2+ efflux, and slightly accelerated 86Rb+ efflux from islet cells. These latter effects were abolished by omission of extracellular Ca2+. Qualitatively similar changes were observed at 15 mM glucose, whereas CGP 28392 was ineffective at 3 mM glucose. These results strongly suggest that an influx of Ca2+ contributes to the slow waves of membrane potential triggered by glucose, and underline the importance of this influx of Ca2+ for the control of insulin release by the sugar.

Cyclic Adenosine-monophosphate Differently Affects the Response of Mouse Pancreatic Beta-cells To Various Amino-acids

1. The membrane potential of mouse beta‐cells was measured in parallel with 86Rb+ efflux and insulin release from mouse islets during stimulation by three types of amino acids and modulation of their effects by glucose and cyclic adenosine monophosphate (cyclic AMP) (forskolin being used to activate the adenylate cyclase). 2. In the absence of glucose, alanine and arginine accelerated 86Rb+ efflux, whereas leucine decreased it. They all depolarized the beta‐cell membrane and slightly increased insulin release. Forskolin had little effect on 86Rb+ efflux, consistently potentiated insulin release but induced electrical activity only in the presence of leucine. 3. The effects of the three amino acids on 86Rb+ efflux and beta‐cell membrane potential were not qualitatively altered by a non‐stimulatory concentration of glucose (3 mM). However, the release of insulin induced by leucine alone or with forskolin was markedly amplified, in contrast to that of alanine or arginine, which was inhibited. 4. In the presence of a threshold concentration of glucose (7 mM), the three amino acids accelerated 86Rb+ efflux and depolarized the beta‐cell membrane. With alanine and arginine, spike activity was transiently observed and coincided with a short‐lived increase in insulin release. With leucine, slow waves with superimposed bursts of spikes occurred and were accompanied by a sustained release of insulin. Forskolin alone also triggered slow waves and bursts of spikes, and increased insulin release. Both effects were larger in the presence of arginine, but not in the presence of alanine. Forskolin considerably increased the electrical and secretory effects of leucine. 5. A higher concentration of glucose (10 mM) induced slow waves with bursts of spikes in all cells and stimulated insulin release. Alanine, arginine and leucine increased 86Rb+ efflux, electrical activity and insulin release. However, the changes produced by the three amino acids displayed different time course, amplitude and characteristics. Forskolin potentiated insulin release and electrical activity induced by glucose alone. These effects were not augmented by alanine, but markedly amplified by arginine or leucine. 6. Several conclusions can be drawn from this study. The three types of amino acids depolarize the beta‐cell membrane by different mechanisms and produce distinct patterns of electrical activity. Slow waves with bursts of spikes occur only if a decrease in K+ permeability contributes to the depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Sparteine Increases Insulin Release By Decreasing the K+ Permeability of the B-cell Membrane

The effects of sparteine on the pancreatic B-cell function have been studied with mouse islets. In the presence of a non-stimulatory concentration of glucose (3 mM), sparteine (0.2-1 mM) decreased the rate of 86Rb+ efflux from islet cells, depolarized the B-cell membrane, induced a glucose-like electrical activity and stimulated insulin release. This increase in release was observed over a large range of glucose concentrations (3-20 mM), and was most marked in the presence of 10 mM glucose. At this concentration of glucose, the effect of sparteine was already detected with 0.02 mM and was maximal with 0.5 mM. Higher concentrations of sparteine only had a transient effect on insulin release. In the presence of 10 mM glucose, 0.2 mM sparteine decreased 86Rb+ efflux and increased 45Ca2+ efflux from islet cells. The effect on 86Rb+ efflux was only transient in the presence of extracellular calcium, whereas the effect on 45Ca2+ efflux required the presence of extracellular calcium. The electrical activity induced by glucose in B-cells was augmented by sparteine which, at a concentration of 0.5 mM, produced a persistent depolarization with continuous spike activity. The potentiation of insulin release by sparteine was not reversible, but was inhibited by adrenaline and completely blocked by omission of extracellular calcium. Sparteine reversed the increase in 86Rb+ efflux and the decrease in insulin release caused by diazoxide. These results show that sparteine increases insulin release by reducing the K+-permeability of the B-cell membrane.

Valinomycin inhibition of insulin release and alteration of the electrical properties of pancreatic B cells

The effects of valinomycin on insulin release and rubidium efflux from perifused isolated rat islets were investigated and correlated with its effects on the electrical properties of mouse B cells studied with microelectrode techniques. Valinomycin produced a (1 · 10−9–1 · 10−6 M) dose- and time-dependent inhibition of (10–15 mM) glucose-stimulated insulin release but did not affect basal secretion. This inhibitory effect rapidly followed addition of the ionophore and equally affected the two phases of glucose-stimulated secretion. It was not reversible by simple washing of the islets, but could be reversed transiently by tetraethylammonium or high extracellular potassium ion levels. At low or high glucose, valinomycin rapidly augmented the rate of 86Rb+ efflux from preloaded islets. Amplitude and rapidity of this effect were dose-dependent and it was antagonized by tetraethylammonium. Glucose metabolism by islet cells was reduced only slightly (15%) by 1 · 10−7 M valinomycin. During the first 6 to 8 min of valinomycin addition, the membrane potential of B cells augmented slowly but the typical bursts of spikes disappeared rapidly. Later on, B cells hyperpolarized more quickly to a stable value of approx. −70 mV. Increasing extracellular K+ immediately depolarized B cells and the linear relationship found between the logarithm of K+ concentration and the membrane potential was characterized by a slope of 58 mV for a ten-fold increase in extracellular K+. These results suggest that valinomycin interferes with the insulin releasing effect of glucose by increasing the potassium permeability of the B cell membrane.

The effects of cesium chloride on insulin release, ionic fluxes and membrane potential in pancreatic B-cells

Cs+ decreases K+ permeability in nerve and muscle cells. Its effects on the pancreatic B-cell function were studied with mouse islets. In the presence of 3 mM glucose, Cs+ substitution for K+ steadily inhibited 86Rb+ efflux and hyperpolarized the B-cell membrane. Addition of Cs+ to a K+-medium also inhibited 86Rb+ efflux, but depolarized the B-cell membrane. None of these changes altered insulin release. Substitution of Cs+ for K+ in a medium containing 10 mM glucose caused a Ca2+-dependent stimulation of insulin release and 45Ca2+ efflux, produced an initial fall and a secondary rise in 86Rb+ efflux and augmented the electrical activity in B-cells. Reintroduction of K+ to the medium was followed by a marked and transient inhibition of insulin release, that was blocked by ouabain and accompanied by an inhibition of 45Ca2+ and 86Rb+ efflux and by a hyperpolarization of the B-cell membrane. Addition of Cs+ to a K+ medium containing 10 mM glucose stimulated insulin release, 45Ca2+ efflux and 86Rb+ efflux. It also increased the electrical activity in B-cells. In the absence of Ca2+, however, Cs+ addition decreased the rate of 86Rb+ efflux. The effects of Cs+ on the B-cell function may be explained by its ability to decrease K+ permeability of the plasma membrane, by its inability to activate the sodium pump, and by a third unidentified effect likely brought about by the accumulation of intracellular Cs+.

Forskolin Suppresses the Slow Cyclic Variations of Glucose-induced Electrical-activity in Pancreatic B-cells

The membrane potential of mouse pancreatic B cells was recorded with microelectrodes. In certain cells, both the slow waves of depolarization and the intervals of repolarization triggered by glucose (10 or 15 mM) displayed regular oscillations in their duration, though the concentration of the sugar remained constant. When forskolin (0.2 microM), an activator of adenylate cyclase, was added to the medium, the electrical activity rapidly became very regular, with slow waves and intervals of constant duration. This effect was unrelated to the overall increase in activity also brought about by forskolin. The oscillations resumed in 75% of the cells after withdrawal of the drug. Under similar conditions, forskolin rapidly and reversibly raised the cAMP concentration in the islets. The data suggest that cAMP is an important modulator of the electrical activity triggered by glucose in insulin-secreting cells.